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Download this complete Project material titled; An Evaluation Of The Aphrodisiac And Fertility Enhancing Effect Of The Methanolic Root Extract Of Cissus Populnea Guill.And Perr. (Vitaceae)with abstract, chapters 1-5, references, and questionnaire. Preview Abstract or chapter one below

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ABSTRACT

Aphrodisiac activity of Cissus populnea was assessed using physical, biochemical and psychological methods. Rats were randomized into five groups of six rats. The first group received normal saline (10ml/kg), the second, third and fourth groups received extract doses of 250mg/kg, 500mg/kg and 1000mg/kg respectively while the fifth group received 5mg/kg of sildenafil citrate orally for 28 days. On the 24th day, the effect of the root extract of Cissus populnea (CPRE) on mounting and mating frequencies was evaluated. The psychological (mood) effect of CPRE was evaluated on the 27th day by exposing the treated rats to the elevated plus maze (EPM) for five (5) minutes and the time spent in the open and close arms of the EPM were recorded. On the 29th day, the rats were anaesthetized with diethylether and blood samples were collected by cardiac puncture for biochemical analysis (testosterone, cortisol, prostate specific antigen (PSA) levels, liver and kidney function parameters). Testes were removed and an incision was made at the caudal epididymis were transferred into a petri dish containing normal saline from where sperm samples were collected using a Neubauler ruled chamber to ascertain the sperm count and morphology. The antioxidant effect of CPRE was compared with that of ascorbic acid using DPPH (2, 2- diphenyl-2- picrylhydrazyl) assay method and was measured at 518nm and the percentage antioxidant activity (AA %) was then calculated.
The acute toxicity study revealed that CPRE was relatively safe. The LD50 value was found to be above 5000mg/kg. The phytochemical screening revealed the presence of saponins, flavonoids, terpernoids and tannins. CPRE and sildenafil citrate significantly increased mounting frequency (P≤0.05) and mating frequency (P≤0.05) respectively compared to the negative control; maximum effect was observed at the dose of 500mg/kg of the extract. There was significant (P≤0.01) and dose-dependent decrease in sperm count in the extract treated rats. The motility and percentage of abnormal sperm cells also decreased in the extract treated
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rats compared to the negative control and standard drug sildenafil citrate. The extract produced significant (P≤0.001) and dose dependent increasein testosterone level compared to the control. Significant increase (P≤0.05) in cortisol level was observed in the sildenafil and extract (250 and 500mg/kg) treated rats compared to the control. There was no significant change in prostate specific antigen level in the CPRE however sildenafil citrate treated rats showed significant ((P0.05) increase in PSA level compared to the control group. The administration of the extract did not show any increase in the level of hepatic andnephrotic enzymes. However there was significant increase in AST level in the extract and sildenafil treated rates compared to the control group. The percentage antioxidant activity of ascorbic acid was significantly (P≤0.05) higher than that of the extract. The extract and sildenafil significantly (P≤0.01) increased the time spent in the open arm while significantly (P≤0.01) decreasing the time spent in the closed arm in the EPM compared to the control group. The methanolic root extract of Cissus populneais relatively safe. The aphrodisiac property may be attributed to a combination of increased in testosterone level, phytochemicals such as saponins and flavonoids and its potential to relieve stress and anxiety. The extract does not have fertility enhancing effect as shown by the decrease in sperm count, motility and increase in percentage of abnormal sperm cell.

 

 

TABLE OF CONTENTS

Cover page Page Title page i Declaration iii Acknowledgement vi Abstract vii Table of Content ix List of figure xiii Abbreviation xv ChapterOne 1.0 Introduction 1 1.1 Statement of problem 2 1.2 Justification 4 1.3 Theoretical frame work 5 1.4 Aims and objectives 9 1.4.1 Specific objectives 10 1.5. Research hypothesis 10 Chapter Two 2.0. Literature review 11 2.1. Etiology of aphrodisiac 11 2.1.1.Classification of aphrodisiac 13 2.2 Male reproductive organ 13 2.2.1Functions of male reproductive organ 14 2.3. Types of sexual disorders 15 2.4. Causes of sexual dysfunction and infertility 218 2.5. Diagnosis of sexual dysfunction 23
2.6. Management of sexual disorders 25
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2.6.1.Life style changes management approach 25 2.6.2. Therapeutic management approach 25 2.7. Role of medicinal plants in the treatment of sexual dysfunction 29 2.8. Plant description 30 2.9. Ethno medical benefits Cissus populnea in disease condition 30 Chapter Three 3.0. Materials and method 33 3.1. Collection of plant material 33 3.2. Extract preparation 33 3.3. Animals 33 3.4. Chemicals and drugs 34 3.5. Phytochemical screening 34 3.6. Acute toxicity studies 34 3.7 Pharmacological screening 35 3.7.1 Experimental design 35 3.7.2 Physical method of assessing plants with aphrodisiac or fertility effects 35 3.7.2.1 Mounting frequency test 35 3.7.2.2. Mating frequency test 36 3.7.3 Biochemical method of assessing plants with aphrodisiac or fertility effects 36 3.7.3.1 Collection and separation ofsera samples for biochemical analyses 36 3.7.3.2 Sperm count and analysis 36 3.7.3.3 Corticosteroid assay 37 3.7.3.4Prostate specific antigen assay 37 3.7.3.5Testosterone 37 3.7.3.6DPPH assay 48 3.7.3.7 Liver and kidney function test 39 3.7.3Psychological method of assessing plants with aphrodisiac or fertility effects 39 3.7.3.1 Anxiety studies 39 3.7.3.2. Learning and memory studies 40
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3.8 Statistical analysis 40 Chapter Four 4.0 Results 41 4.1 Extract yield 41 4.2 Phytochemical constituents 41 4.3 Acute toxicity studies effect of Cissus populnea root extract 42 4.4 Effect of Cissus populnea root- extract on physical sexual behavior 43 4.4.1 Male mounting frequency test 43 4.4.2 Male mating frequency test (sexual episode) 44 4.5 Biochemical effect of the extract of Cissus populnea root 45 4.5.1 Effect of extract on sperm count 45 4.5.1.1. Sperm morphology and motility 46 4.5.2 Testosterone assay of the effect of Cissus populnea root extract 47 4.5.3 Cortisol assay of the effects of Cissus populnea root extract 48 4.5.4 Prostate specific antigen assay of the effect of Cissus populnea root extract 49 4.5.5 Antioxidant effect of the extract of Cissus populnea root 50 4.6.1 Liver function monitoring parameters 51 4.6.2 Kidney function monitoring parameters 57 4.7 Psychological effect of Cissus populnea root extract 64 4.7.1 Effect of the extract of Cissus populnea root on anxiety 64 4.7.2 Effect of the extract of Cissus populnea on memory 65 Chapter Five 5.0 Discussion 66 Chapter Six 6.0 Summary, Conclusion and Recommendation 72 6.1 Conclusion 72
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6.2 Recommendation 72 REFERENCE 73 APPENDIX 90
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CHAPTER ONE

1.0 INTRODUCTION Aphrodisiac refers to any substance that increases sexual desire arousal and performance. Their use dates back to millennia (Sandroni, 2001). They may be of plant or animal sources and can be classified based on those that increase desire and arousal, enhance or improve penile erection and those that enhance sexual pleasure. The use of aphrodisiacs is informed by the needs of the client to ameliorate a sexual disorder or dysfunction. Sexual disorders may be classified into sexual desire disorders, sexual arousal disorders, orgasmic disorders and sexual pain disorders (Hatzimouratidis and Hatzichristou, 2007). These classifications are based on International Classification of Diseases, Diagnostic and Statistical Manual of Mental Disorder-IV, The National Institute of Health Consensus Conference on impotence. Sexual dysfunction is the inability to enjoy sexual intercourse. The most occurring form of male sexual dysfunction is erectile dysfunction (ED), while data for the most occurring female sexual dysfunction (FSD) is inconsistent since sexual function involves desire, arousal and gratification (Sarit and Goldberg, 2009). Sexual activity is essential for procreation and general wellbeing as it bonds a relationship and has a calming effect. Sex involves the psychosocial and physiological activities to maintain a normal sexual function. Endocrine disorders or imbalance (Araujo et al., 2000), life style patterns and substance abuse like smoking, alcohol consumption (Hammedeh et al., 2010), psychological stress and neurological diseases, cardiovascular diseases (Adegite et al., 2009), penile diseases and surgery and age affect sexual activity.
It takes different forms in men and women and may be acute or situational due to response to environment, loss of loved one or job. It may be persistent or chronic due to an underlying disease conditions. Whatever form it takes, sexual dysfunction takes a toll on the relationship and general wellbeing of the person. Most sexual dysfunction may be due to manageable
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health problems. This is a concern in developing countries where access to health facilities is a challenge. Most men and women may not have the right diagnosis and poor management of a disease condition may worsen the existing situation. 1.1 Statement of Research Despite advances in medical science and the discovery of the block buster aphrodisiac (sildenafil citrate), Viagra®, sexual dysfunction in men and women is still a major public health issue (Shiri et al., 2005; Dzeladuin et al., 2009). Men are more outspoken to physicians on sexual dysfunction while most women keep their concerns private. The World Health Organization (WHO) projected that, in 2025 about 322 million males will be sexually dysfunctional (Araujo et al., 2000). The discovery of the phosphodiesterase Vinhibitors, increased the awareness of sexual dysfunction globally. The prevalence of men sexual dysfunction is 56% in the United States of America while that of women is 63%. Prior to this development there were no data on female sexual dysfunction. Thirty-four (34%) of men between 40-70 years have one form of sexual dysfunction (Oksuz and Malhan, 2005). In a study to ascertain the prevalence of sexual dysfunction in male above 35 years in three countries, 80.8% of the study group in Pakistan had sexual dysfunction while, 57% of the men in Sub-Saharan Africa were reported to have the same sexual dysfunction (Sheer et al., 2004).
In Nigeria, the prevalence of sexual dysfunction is 57.4% among men above 35 years (Sheer et al., 2004). Fatusi et al., (2003), reported prevalence of sexual dysfunction of 38.5% and 63.9% among males between the ages of 31-40 and 61-70years respectively in South Western Nigeria. Adegite et al. (2009), also reported that, sexual dysfunction has a prevalence of 58% among males with diabetes in Nigeria. Prevalence and incidence studies of female sexual dysfunction are confounded by lack of consistent methodology. The role of cultural belief
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and challenge in measuring quantitative and qualitative sexual function complicates the classification and development of concise models for female sexual dysfunction (Sarit et al., 2009). Reproductive and sexual dysfunction are more pronounced with increase in age (Simeon et al., 2004). The inability to maintain a healthy sexual and reproductive life leads to depression, nervousness, anxiety, fear and ultimately low quality of life. About 12,000 divorce cases occured annually in Nigeria since 1975 (Almanac book of facts, 1977). Alice (1996), attributed 68.9% of divorce to the inability of one partner to satisfy the other and prolonged lack of conception. Infertility among males between the ages of 35-50 years is attributed to low sperm count, low sperm motility and abnormal sperm cells (Nwafia et al, 2006). According to (Ozoemena et al., 2011), hormonal imbalance is responsible for 70% of infertile couples between the ages of 36-55 years.
In spite of these prevalence data, orthodox medical approach and research has not provided the desired treatment for the various types of sexual and reproductive dysfunction. The available drugs such as hormone replacement therapy (testosterone, estrogen, and progesterone), serotonin re-uptake inhibitors (clomipramine, fluoxetine), alpha blockers, vasodilators, phosphodiesterase V inhibitor and procedures like the use of prosthetics are expensive, not readily available and may require routine clinical checkup and monitoring which may be lacking in developing countries. The side effects of some of these agents pose a challenge in the management of existing health condition or predisposes the patient to some other health conditions. Hormone replacement therapy is associated with hepatotoxicity and hypertension while alpha blockers are associated with priapism and arrhythmia. Vasodilators can cause priapism and hypotension, with prostaglandins causing pain at injection site. Some patients may require drugs to manage sexual dysfunction all through life while they are on medications for other medical conditions that also require life-long drugs. The burden on the National Health Insurance Scheme, an already inadequate overwhelmed medical system and
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the cost of living on family and friend calls for more effective drugs in the management of disease conditions. In view of these challenges, it is pertinent that researchers and pharmaceutical companies chart a global approach to provide safe, effective, available and affordable therapeutic agents for the therapeutic management of sexual and reproductive dysfunction with the aim to reduce the incidence and prevalence of sexual dysfunction, improving sexual function and general wellbeing. This study seeks to evaluate the aphrodisiac and fertility-enhancing effects of the methanolic extract of Cissus populnea root. 1.2 Justification The incidence of erectile dysfunction is on the increase. The worldwide prevalence was 152 million in 1995 and projected to be 322 million by 2025 (Aytac et al, 1999). This increase may be due to substance abuse for sexual pleasure or socialization. Women increasingly have a voice to say how they feel and see themselves as equal to men. The men are therefore, pressured to satisfy their partners despite declining hormonal levels with increase in age. The intense search for medicinal plants that are cost effective, relatively safe and accessible may be due to the burden of the health condition. Sexual dysfunction is more prevalent in patients between 40 – 70 years, which coincidentally is also the period in a man‟s life that age predisposes him to cardiovascular diseases and endocrine disorders like diabetes that may cause sexual disorders. The prevalence of disease conditions like hypertension and diabetes is on the increase globally (American Heart Society, 2011).
Male sexual dysfunction is more prevalent due to the fact that it is most reported and the decline in male hormone naturally predisposes men to sexual dysfunction therefore, the need to focus more on it. The stigma associated with male sexual dysfunction and the resultant
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effect on his productivity, self-esteem and the inability to maintain a healthy relationship make it a public health issue. Increasing research into medicinal plant may be informed by the fact that, many people with sexual dysfunction in developing countries do not have access to medical facilities and the need for drugs that are readily available, affordable and have less deleterious side effects. Medicinal plant that have been reported to have aphrodisiac and fertility enhancing properties include: Ambergris, Tribulus terrestriss, Tunera diffusa and Epimedium grandiflorum. In Nigeria, plant parts like Garcina kola, Carica papaya are used to treat sexual disorders. There is need for effective corroboration of traditional medicine practice with scientific approach on information on collection, preparation, side effects, efficacy, safety and standardization of some of the plant parts. The folklore claims of some medicinal plant were unfound when subjected to scientific evaluation. Hence, the need to study more plants which are sources of new drugs. 1.3 Theoretical Frame Work 1.3.1 Acute toxicity study
Acute toxicity study in animals is usually the first test that is performed on any compound or pharmaceuticals intended for human use. It evaluates adverse effects produced by the test compound as a result of either a single exposure or multiple exposures within a short period of time (typically not exceeding 24 hours). Information obtained from these studies is therefore useful in identifying doses that cause no adverse effects; dose determination in animal efficacy and repeat dose studies and major (life-threatening) toxicity. It gives an insight into the mechanism of biological/toxic effects and the median lethal dose (LD50) that provides many indices of potential types of drug activity. It may also aid in selection of starting doses for Phase 1 human studies and provide information relevant to acute
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overdosing in humans. The most common conventional methods used for carrying out acute toxicity studies include the one described by Lorke‟s (1983), OECD 423 guidelines (2001) and acute toxic class (2002). They all have the advantage of using only a few animals to achieve all the objectives of acute toxicity studies. In this study, the OECD 423 guideline (2001) was used. 1.3.2 Animal models for studying aphrodisiac activity of drugs and medicinal plants. Sexual dysfunction, a major cause of couple‟s infertility and lack of sexual activity is more prevalent in males than females thus the focus on male sexual difficulties (Yakubu et al., 2007). An aphrodisiac is defined as any drug or food that arouses sexual instinct, induces venerably desire and increases pleasure and performance. Several invitro and invivo models have been used to investigate the aphrodisiac properties of drugs (Yakubu et al., 2007, Varsha et al., 2013). These can be categorized into physical (behavioral), biochemical and psychological (mood) methods.
1.3.2.1 Physical methods
The physical methods that are used to assess the aphrodisiac effect of a substance include male sexual behavior which uses techniques such as Mount frequency, mount latency, intromission frequency, intromission latency, ejaculation frequency, post ejaculatory interval, index of libido, and computed male sexual behavior parameter. Orientation behavior, determination of hesitation time and attraction towards female, test of potency, test for libido, penile microcirculation and intracavernous pressure studies are also used. (i) Mounting frequency test
Mounting is defined as an expression of sexual desire when one animal climbs another from the posterior with the aim of initiating sexual activity. Mount frequency (MF) is the number of mounts from the time of introduction of the female until ejaculation (Guathman et al., 2002).
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(ii) Mating frequency test Mating or intromission is the introduction of the reproductive organ of a sexual partner into the other partner during a sexual activity e.g. the penis into the vagina. The intromission frequency (IF) is the number of intromission from the time of introduction of female until ejaculation. Any medicinal plant with aphrodisiac property will produce statistically significant increase in mount and intromission frequencies. These indices indicate sexual arousability, motivation and vigour. 1.3.2.2Biochemical methods (i)Sperm analysis Fertilization requires adequate and normal sperm count, morphology and activity to occur. The most important male infertility test is a semen analysis (sperm evaluation) as more than 90% of male infertility cases are due to low sperm counts, poor sperm quality or both. Sperm analysis will provide information on the effect of the plant on sperm count, morphology and activity indicating fertility enhancing effect or otherwise. (ii)Hormonal determination (a) Testosterone assay Testosterone is the main male hormone responsible for male sexual characters. It also plays a major role in spermatogenesis. Testosterone has been shown to stimulate desire for sex and helps maintain the health of the tissues of the penis enabling erections (Aversa et al., 2000). Increasedlevels of serum testosterone will thus, be considered as evidence of aphrodisiac property while a decreased level will be considered as lack of aphrodisiac property. It may also indicate possible mechanism of aphrodisiac action. (b)Corticosteroids (cortisol) assay
Cortisol, a hormone produced by the hypothalamic pituitary adrenal (HPA) axis is released in response to oxidative stress (Sikka and Wang, 2008). HPA also controls spermatogenesis.
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Acute psychosocial stress has been shown to potentiate the secretion of cortisol (Biondi and Picardi, 1999). A blood cortisol level test can thus serve as a stress barometer. An elevated circulating cortisol level (which promotes the production of reactive oxygen species (ROS)) has been reported to cause gonadal and sexual dysfunction in males. Information obtained from the study will indicate the effect of the extract on blood cortisol level and its consequences on male reproductive activities. (c) Prostate specific antigen (PSA) assay Prostate specific antigen (PSA) is secreted by the prostate gland to liquefy the semen produced by the prostate. It thus enables sperm cells to swim freely to fertilize the egg (Anitha et al., 2009). An elevated serum PSA level has been reported to indicate prostate disorders which have been linked with sexual apathy and semen clotting. Information obtained from the study will indicate the effect of the extract on serum PSA level and the implication on sexual and reproductive activities in males. PSA level may increase as a result of inflammation of prostate gland (prostatitis) or prostate cancer. An injury or sexual activity may also briefly raise PSA levels. PSA concentration is a marker of prostatic disease and inflammation. (iii) DPPH antioxidant assay
The human body uses an antioxidant defense system to neutralize excessive levels of reactive oxygen species (ROS) that have been associated with degenerative diseases. Antioxidant scavenges these free radicals from the system and protects germ and sertoli cells which support spermatogenesis (Madhusudana et al., 1997). 1, 1, diphenyl-2-picryl hydrazyl (DPPH) assay is widely used to determine free radical scavenging activity of plant samples (Kato et al, 1988, Kumarasami et al., 2007). The scavenging effect of the plant sample will be compared with that of a standard antioxidant (ascorbic acid). The result will provide information on the antioxidant properties of the drug.
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1.3.2.3Psychological (mood) method. Male sexual dysfunction is caused by several factors including psychological disorders (performance anxiety, strained relationship, depression, stress, guilt and fear of sexual failure). Therefore, any stimuli that result to a psychological burden may be associated with anxiety and may causes sexual dysfunction. (i)Elevated plus maze test Anxiety, a state of excessive fear is characterized by motor tension, sympathetic hyperactivity, apprehension and vigilance syndromes. The elevated plus maze (EPM) model, is one of the most validated tests for assaying anxiolytic substances (Pellow and File, 1986). The fear due to height induces anxiety in rats. The time spent in the open and close arms of EPM provides information on the anxiolytic or anxiogenic effect of the drug. This test will provide information on the effect of the plant extract on anxiety. Lack of cognition may be due to disease condition which may lead to sexual challenges. It may be due to disease that affect the central nervous system. The exposure of the rats to the EPM on the 7th, 14th and 21st days will provide information on the effect of the drug on cognition enhancement which is key in ascertaining the effect of the extract in memory enhancement and alertness. Increased time spent in the open arm implies the drug has effect on memory while the decreased time spent in the open arm will be considered as lack of effect on memory. 1.4 Aim and Objectives The aim of this study is to evaluate the aphrodisiac and fertility enhancing properties of the methanolic root extract of Cissus populnea in wistar rats.
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1.4.1 Specific objectives The specific objectives of the study are to:
(i) Carryout acute toxicity study of the methanolic root extract of Cissus populnea to guide in the dose selection for efficacy studies.
(ii) Evaluate the effect of the methanolic root extract of Cissus populnea on sexual behavior (mounting and mating frequencies).
(iii) Evaluate the effect of the methanolic root extract of Cissus populanea on biochemical parameters (sperm count, sperm morphology, testosterone, cortisol, prostate specific antigen levels, DPPH antioxidant assay, liver and kidney function).
(iv) Evaluate the psychological (mood) effect of the methanolic root extract of Cissus populnea.
1.5 Research Hypothesis The methanolic root extractCissus populneahas no aphrodisiac and fertility-enhancing properties.
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